-Histofine® is NICHIREI BIOSCIENCES INC.’s registered trademark. Contact us for details. 02/2010

-Histofine®

DAB-3S kit Peroxidase Chromogen/Substrate kit

500 tests Code: 415192F 1500 tests Code: 415194F

3, 3’-DIAMINOBENZIDINE TETRAHYDROCHLORIDE (DAB) SUBSTRATE KIT

SUBSTRATE FOR HORSERADISH PEROXIDASE INTENDED USE For In Vitro Diagnostic Use. For professional users． Diagnosis should be made based on the clinical history and results of diagnostic tests of the patient. FORM Liquid. This product consists of followings: [500 tests] [1500 tests] Reagent A: DAB solution concentrate … 3 ml×1 bottle 9 ml×1 bottle Reagent B: Enhancer solution concentrate … 3 ml×1 bottle 9 ml×1 bottle Reagent C: Hydrogen peroxide solution concentrate … 3 ml×1 bottle 9 ml×1 bottle Dropping bottle … 1 bottle 1 bottle Tube … 1 tube 1 tube DESCRIPTION

-Histofine® DAB-3S kit is the chromogen/substrate system suitable for use in peroxidase-based immunohistochemical staining. DAB may produce brown precipitates at the site of the target antigen or nucleic acid reacting with peroxidase. PREPARATION of Chromogen/Substrate solution 1. Add 1 drop of Reagent A (DAB solution concentrate) to 1 ml of distilled water in the tube, and mix well. 2. Add 1 drop of Reagent B (Enhancer solution concentrate) to 1 ml of above diluted Reagent A solution, and mix

well. 3. Add 1drop of Reagent C (Hydrogen peroxide solution concentrate) to 1 ml of above mixture solution of Reagent A

and Reagent B, and mix well as final mixture*1. *1: Final mixture is stable for 14 days at 2-8°C under the light shielded condition.

Precipitates may be produced with time. The precipitates adhered on slides or tissues will not affect on the staining, however, in some cases, obtaining clear image may not be suitable. To avoid such adhesion, following procedures are recommended.

1. Use supernatant solution of the mixture. 2. Rinse the slides in PBS containing 0.1% Tween 20 or TBS containing Tween 20 at room temperature for 3 minutes, at

respective steps of before and after the staining described in USAGE. USAGE Adjust the final mixture to room temperature prior to use. React the objective tissue section with peroxidase conjugate in advance. At the same time, staining of Negative control slide using PBS in place of primary antibody is recommended to be prepared for checking the staining result. For color development, incubate the tissue section on the slide with 1-2 drops of the final mixture using Dropping bottle*2 at room temperature for 5-20 minutes. Adjust the reaction time by microscopic observation by contrast with the Negative control slide. After adequate coloration, wash the slide with tap water. Mount the slide with permanent mounting media and fix with cover slip after washing, dehydrated in graded series of alcohol and cleared in xylene. *2: 1) Dropping bottle is used for dropping the final mixture, not for mixing or storage of reagents.

2) When the final mixture is removed from tube to the dropping bottle, adequate pipetting is required. 3) Rinse the dropping bottle thoroughly with distilled water and dry after use.

English

-Histofine® is NICHIREI BIOSCIENCES INC.’s registered trademark. Contact us for details. 02/2010

NICHIREI BIOSCIENCES INC. 6-19-20, Tsukiji, Chuo-ku, Tokyo, 104-8402, JAPAN Phone:81-3-3248-2207, Facsimile:81-3-3248-2243

STORAGE Store at 2-8°C. PRECAUTIONS 1. Avoid contact with eyes, skin and clothing as well as ingestion and inhalation. 2. Handle Reagent A with adequate attention due to its DAB may evolve into mutagens. 3. Handle Reagent C with adequate attention due to hydrogen peroxide solution contained. 4. Dispose Reagent A including prepared solutions with DAB as well as unused solutions and components as

hazardous chemical in accordance with all applicable rules and regulations.

EXBIO Olomouc s.r.o. Ovesna 14, 779 00 OLOMOUC CZECH REPUBLIC